Derivatives of Pyrrolo-Pyrazines Having a Kinase Inhibitory Activity and Their Biological Applications

ABSTRACT

The invention relates to pyrrolo[2,3b]-pyrazine derivatives having the general Formula (I) wherein R2 and R3 are identical or different and represent H, C1-C6 alkyl, said alkyl being a straight or branched-chain alkyl, which can be substituted, R6 is an optionally substituted aromatic cycle Ar or a cycloalkyl, said cycloalkyl being optionally substituted by an aryl group which can also be substituted, R7 is H, C1-C6 alkyl, (alk.)n-hal., CH2—CH═CH2, CH2-cycloalkyl, CH2—Ar, with “alk.” being a C1-C6 alkylene group, n being 1-6, Z is H or CH3. Application as active principle of pharmaceutical compositions, particularly for treating or preventing neurodegenerative disorders and proliferative disorders.

The invention relates to derivatives of pyrrolo-pyrazines having akinase inhibitory activity and their biological applications. Proteinkinases catalyse the phosphorylation of serine, threonine and tyrosineresidues of proteins, using ATP or GTP as the phosphate donor. Proteinphosphorylation is considered as one of the main post-translationalmechanisms used by cells to finely tune their metabolic and regulatorypathways.

Protein kinases (an estimated 800 in the human genome), and theircounterparts the protein phosphatases, appear to be involved in mosthuman diseases. This is the reason why screening for potent andselective inhibitors of protein kinases has intensified over the lastfew years.

The inventors have focused their efforts on two families of kinases,cyclin-dependent kinases (CDKs) and glycogen synthase kinase-3 (GSK-3).

CDKs are involved in controlling the cell cycle apoptosis, neuronalfunctions and neurodegeneration, transcription and exocytosis.

GSK-3, an essential element of the WNT signaling pathway, is involved inmultiple physiological processes including cell cycle regulation bycontrolling the levels of cyclin Dl and β-catenin, dorso-ventralpatterning during development, insulin action on glycogen synthesis,axonal outgrowth, HIV-1 Tat-mediated neurotoxicity, and phosphorylationof tau, a characteristic of Alzheimer's disease. Applications ofCDK/GSK-3 inhibitors are being evaluated against cancers,neurodegenerative disorders such as Alzheimer's disease, diabetes,proliferation of protozoan parasites and viral infections (HIV,cytomegalovirus and herpes virus) (1).

CDK inhibitors include the purines olomoucine, roscovitine, purvalanols,CVT-313, C2-alkylynated purines, H717 and NU2058, piperidine-substitutedpurines, toyocamycin, flavopiridol, indirubins, paullones,γ-butyrolactone, hymenialdisine, indenopyrazoles, the pyrimidines NU6027and CGP60474, pyridopyrimidine, the aminopyrimidine PNU 112455A,oxindoles, PD0183812, cinnamaldehydes, quinazolines, fasclaplysin,SU9516 and benzocarbazoles (reviewed in ref. 1, 2-8). GSK-3 inhibitorsinclude indirubins, paullones, maleimides and lithium.

The inventors have now identified a new family of kinase inhibitorsselective for CDK1/2/5 and GSK-3α/β, acting in the sub-micromolar rangeby competing with ATP for binding to the kinase active site, as revealedby enzymological studies and crystal structure studies.

Said family has a therapeutical value in pathological situationsinvolving CDKs and/or GSK-3α/β deregulations.

The invention thus relates to novel derivatives of pyrrolo-pyrazines.

It also relates to a method for preparing said derivatives.

According to still another aspect, the invention relates to the use ofsaid derivatives as active principle of drugs.

The pyrrolo[2,3b]-pyrazine derivatives of the invention have the generalformula (I):

wherein

-   -   R2 and R3 are identical or different and represent H, C1-C6        alkyl, said alkyl being a straight or branched-chain alkyl,        which can be substituted,    -   R6 is an optionally substituted aromatic cycle Ar or a        cycloalkyl, said cycloalkyl being optionally substituted by an        aryl group which can also be substituted,    -   R7 is H, C1-C6 alkyl, (alk.)_(n)-hal., CH₂—CH═CH₂,        CH₂-cycloalkyl, CH₂—Ar,    -   Z is H or CH₃.        Preferably, R2 and R3, and/or Z and/or R7 are different from H.

Ar is preferably phenyl, naphtyl, furyl, thienyl, pyridyl, cyclopropylphenyl, phenyl dioxolyl.

“Cycloalkyl” is a C3-C6 cycloalkyl.

Substitutions of the alkyl group, aromatic cycle or cycloalkyl areselected in the group comprising one or more halogen (F, Cl, Br, I,CF₃), OH, NH₂, N(H, alkyl); N(alkyl)₂, O-alkyl, COOH, COO-alkyl, CONH₂,CON(H,alkyl), CON(alkyl)₂, NHCONH₂, NHCON(H,alkyl), NHCON(alkyl)₂,N(alkyl) CONH₂, N(alkyl)CON(H,alkyl), N(alkyl)CON(alkyl)₂, alkoxy, CN,O—SO₂—NH₂, O—SO₂—N(H,alkyl), —O—SO₂—N (alkyl)₂, SH,S-alkyl. One or moresubstituents can be present.

“Alkyl” is a C1-C6 alkyl and includes isomers.

“Alkoxy” has a C1-C6 alkyl group.

“Alk.” is a C1-C6 alkylene group, n is 1-6, and “hal.” is F, Cl, Br, Ior CF₃.

Said pyrrolo[2,3-b] pyrazines, also designated aloisines hereinafter,are potent kinase inhibitory scaffold, and selective for CDKs andGSK-3α/β, acting for most of them in the sub-micromolar range.

Kinetic studies, as well as the resolution of co-crystal structures ofCDK/aloisines demonstrate that aloisines act by competitive inhibitionof ATP binding to the catalytic subunit of the kinase. They interactwith the ATP-binding pocket through two hydrogen bonds with backbonenitrogen and oxygen atoms of Leu 83.

Said aloisines are also characterized in that they inhibit cellproliferation by arresting cells both in G1 and G2 as illustrated by theExamples hereinafter.

Preferred derivatives of pyrrolo-pyrazines have formula (II):

wherein

-   -   the phenyl group at position 6 is substituted by one, two or        three R substituents selected in the group comprising:    -   H, —OH, alkyl, —O alkyl, hal., —NH₂, —N(H,alkyl), —N(alkyl)₂,        —O—SO₂—NH₂, —O—SO₂—N(H, alkyl), —O—SO₂—N(alkyl)₂, —COOH,        —COO-alkyl, CONH₂, —CON(H,alkyl), —CON(alkyl)₂,    -   R7 is H, alkyl, (alk.)_(n) hal., —CH₂—CH═CH₂,        (alk.)_(n)-cycloalkyl, alk.-Ar, and    -   Z is H or CH₃.        In a preferred group, Z and/or R7 are different from H.

A preferred group of said family has an IC₅₀ value≦10 μM with respect tocorrespond to CDK1/cyclin B, CDK5/p25 and GSK-3. They correspond to thederivatives of formula (II) wherein

R=H, OH, alkoxy, hal., alkyl and R7=H or to derivatives whereinR=alkoxy, and R7=alkyl, (alk.)_(n)-hal., CH₂—CH═CH₂, or

R=O—SO₂—N-(alkyl)₂ preferred, hal., OH, R7=alkyl, n=1-3 and Z=H.

A more preferred group of said family of formula (II) has an IC₅₀value≦5 μM with respect to CDK1/cyclin B, CDK5/p25 and GSK-3.

They correspond to the derivatives of formula (II) wherein R=H,p-alkoxy, p- and m-alkoxy, p-OH, p-hal., p-alkyl, p-O—SO₂—N(alkyl)₂, R7is alkyl, (alk.)_(n)-hal., CH₂—CH═CH₂, or H, Z is H, and n=1-3.

Preferred derivatives of said group correspond to compounds wherein (a-ecorrespond to the position of R on the phenyl group):

-   -   the phenyl group is unsubstituted and R7 is H, or    -   Ra, Rb and Rd=H, Rc=alkoxy, OH or hal., and R7=H, or    -   Ra, Rb and Rd=H, Rc=alkoxy and R7=alkyl, or    -   Ra and Rd=H, Rb and Rc=alkoxy and R7=alkyl, or    -   Ra, Rb and Rd=H, Rc=alkoxy and R7 alkyl, or    -   Ra, Rb and Rd=H, Rc=alkoxy, OH, hal. and R7=alkyl,        (alk.)_(n)-hal, CH₂—CH═CH₂, or    -   Ra, Rb and Rd=H, Rc=OH, R7=alkyl.

A still more preferred group of said family of formula (II) has anIC₅₀≦1 μM with respect to CDK1/cyclin B, CDK5/p25 and GSK-3.

They correspond to derivatives of formula (II) wherein R is p-alkoxy,p-O—SO₂—N-(alkyl)₂, p-OH and R7 is alkyl.

Preferred derivatives correspond to compounds with Ra, Rb and Rd=H,Rc=alkoxy, O—SO₂—N(alkyl)₂, or OH and R7=alkyl.

In a particularly more preferred group, the derivatives have an IC₅₀≦0.5μM with respect to CDK1/cyclin B, CDK5/p25 and GSK-3. Particularlyadvantageous derivatives have Ra, Rb and Rd=H, Rc=alkoxy or OH andR7=alkyl.

Another preferred group of said family of formula (II) has an IC₅₀value≦10 μM with respect to CDK1/cyclin B and CDK5/p25 or GSK-3, or toCDK5/p25 and GSK-3.

The invention particularly relates to the group with derivatives havingan IC₅₀≦10 μM with respect to CDK5/p25 and GSK-3.

In such a group, R=H, OH, alkoxy, hal., alkyl, O—SO₂—N(alkyl)₂, andR7=H, alkyl, (alk.)_(n)-hal., CH₂—CH═CH₂.

Preferred derivatives of said group have an IC₅₀ value≦5 μM with respectto CDK5/p25 and GSK-3. In said derivatives R is H, p-alkoxy, OH, hal.,O—SO₂—N-(alkyl)₂ and R7 is H, alkyl, (alk.)_(n), hal., CH₂—CH═CH₂.

Advantageous derivatives have Ra, Rb, Rc, Rd and R7=H, or Ra, Rb andRd=H, Rc=alkoxy, hal., (alk.)_(n)-hal., or OH and R7=H, or Ra, Rb andRd=H, Rc=alkoxy or OSO₂—N(alkyl)₂, hal., OH and R7=alkyl, or Ra andRd=H, Rb and Rc=alkoxy and R7=alkyl.

More preferred derivatives of said group have an IC₅₀ value≦1 μM withrespect to CDK5/p25 and GSK-3. In such derivatives,

R=p-alkoxy, p- and m-dialkoxy, hal., p-O—SO₂—N(alkyl)₂, p-OH and R7=H oralkyl.

Particularly advantageous derivatives have Ra, Rb, Rd H, Rc=alkoxy andR7=alkyl, or Ra and Rd=H, Rb and Rc=alkoxy and R7=alkyl, or Ra, Rb andRd=H, Rc=O—SO₂—N(alkyl)₂ or OH and R7=alkyl.

Still more preferred derivatives have an IC₅₀≦0.5 μM with respect toCDK5/p25 and GSK-3. Advantageous derivatives have Ra, Rb, and Rd=H,Rc=alkoxy or OH, and R7=alkyl.

In another group of the invention, the, derivatives have an IC₅₀≦10 μMwith respect to CDK1 and GSK3.

In derivatives of said group R=H, OH, alkoxy, hal., alkyl, CN,O—SO₂—N(alkyl)₂ and R7=H, alkyl, (alk.)_(n)-hal, CH₂—CH═CH₂,alk.-cycloalkyl, alk.-aryl.

In a preferred group of said family, the derivatives have an IC₅₀≦5 μMwith respect to CDK1 and GSK-3.

In advantageous derivatives, R=H, p-alkoxy, p- and m-alkoxy, p-OH,p-hal., p-O—SO₂—N(alkyl)₂, p-CN, and R7=H or alkyl, (alk.)_(n) hal.,CH₂—CH═CH₂, (alk.)_(n)-cycloalkyl, (alk.)_(n)-aryl.

Corresponding preferred derivatives have Ra, Rb and Rd=H,

Rc=alkoxy, OH, hal., alkyl, CN and R7=H, or Ra, Rb,

Rd=H,

Rc=alkoxy and R7=alkyl, (alk.)_(n)-hal. or CH₂—CH═CH₂, or Ra and Rd=H,Rb and Rc=alkoxy and R7=alkyl, or Ra, Rb and Rc=H, Rd=O—SO₂—N-(alkyl)₂,and R7=alkyl, or Ra, Rb and Rd=H, Rc=hal. and R7=(alk.)_(n)-aryl.

In a still more preferred group of said family, the derivatives have anIC₅₀ value≦1 μM with respect to CDK1 and GSK-3.

Corresponding derivatives have R=p-alkoxy, p-O—SO₂—N(alkyl)₂, p-hal., H,p-OH, R7=alkyl, or (alk.)_(n)-hal, CH₂—CH CH₂, (alk.)_(n)-cycloalkyl,(alk.)_(n)-aryl.

Preferred derivatives have Ra, Rb and Rd=H, Rc=alkoxy, OH,O—SO₂—N(alkyl)₂, hal. and R7=alkyl, CH₂—CH═CH₂, CH₂-cycloalkyl.

In a particularly more preferred group of said family, the derivativeshave an IC₅₀ value≦0.5 μM with respect to CDK1/cyclin B and GSK-3.

Advantageous derivatives have Ra, Rb and Rd=H, Rc alkoxy or OH andR7=alkyl.

The invention also relates to the group with derivatives having anIC₅₀≦10 μM with respect to CDK1/cyclin B and CDK5/p25.

In such a group, R=H, OH, alkoxy, hal., alkyl, O—SO₂—N(alkyl)₂ and R7=H,alkyl, (alk.)_(n)-hal., CH₂—CH═CH₂.

Preferred derivatives have an IC₅₀≦5 μM with respect to CDK1/cyclin Band GSK-3.

In such derivatives, R is preferably H, O-alkoxy, p-alkoxy, m- andp-alkoxy, p-OH., p-hal., p-O—SO₂—N(alkyl)₂ and R7 is H, alkyl,(alk.)_(n)-hal., CH₂—CH═CH₂.

Particularly advantageous derivatives have Ra, Rb, Rc, Rd and R7=H, orRa=OH and Rb, Rc, Rd and R7=H, or Rc, Rb and Rd=H, Rc=alkoxy, OH or hal.and R7=H, (alk.)_(n)-hal., CH₂—CH═CH₂, alkyl, or Ra and Rd=H, Rb andRc=alkoxy and R7=H, or Ra, Rb and Rd=H, Rc=O—SO₂—N-(alkyl)₂ or hal. andR7=alkyl.

In a still more preferred group, the derivatives have an ICs₅₀≦1 μM withrespect to CDK1/cyclin B and GSK-3.

Advantageous derivatives have R=p-alkoxy, p-O—SO₂—N(alkyl)₂, p-hal.,p-OH and R7=alkyl.

Particularly preferred derivatives have Ra, Rb and Rd=H, Rc=alkoxy OH orO—SO₂—N(alkyl)₂ and R7=alkyl.

In a particularly preferred group the derivatives have an IC₅₀≦0.5μMwith respect to CDK1/cyclin B and GSK-3.

In preferred derivatives, Ra, Rb, and Rd=H, Rc=alkoxy or OH andR7=alkyl.

Another preferred family with an IC₅₀≦10 μM with respect to CDK1/cyclinB, CDK5 and GSK-3 has formula (III), and even ≦5 μM with respect toCDK5/p25 and GSK-3.

Still another preferred family has formula IV with an

IC₅₀≦5 μM with respect to CDK1/cyclin B, CDK5/p25 and GSK-3.

Said derivatives have interestingly an IC₅₀ value≦1 μM with respect toCDK5/p25 and GSK-3.

The invention also relates to a method for preparing said aloisinescomprising reacting alkyl-pyrazines of formula (V):

wherein:

R1 and R3 are as above defined, and Alkyl is a C1-C6 alkyl, witharomatic nitrites, R6CN, wherein R6 is as above defined.

Advantageously, the alkylpyrazine derivatives of formula (V) are addedto an organic solvent containing butyllithium or analog, at atemperature not exceeding 0° C. and preferably of about −40° C. Theresulting solution is stirred during 30 min to about 1 h. The nitrilederivative is then added and the solution is stirred during 30 min toabout 1 h, and further at the ambient (around 20° C.) for about 1 to 20h.

After hydrolysis, the resulting derivative is recovered, purified andcrystallized if desired.

Alkylpyrazines can be obtained by reaction of pyrazinylmethyllithiumwith bromoalcanes, and benzonitriles. Demethylation of methoxy compoundscan be achieved by refluxing in, acidic conditions. The time requiredfor demethylation varied from 3-20 h.

As above mentioned, and as illustrated in the Examples hereinafter, saidcompounds strongly inhibit CDK1 and/or CDK5 and/or GSK-3.

By acting on said kinases, which represent the major kinases involved inthe hyperphosphorylation of substrates in neurodegenerative diseases,said derivatives are of great interest as active principles of drugs forpreventing and treating corresponding conditions. They have also antiproliferative effects.

The invention thus relates to pharmaceutical compositions comprising aneffective amount of at least one derivative as above defined as activeprinciple in association with a pharmaceutically acceptable carrier.

Said carrier may be solid, or liquid, depending on the administrationform.

Said pharmaceutical compositions are useful for treating or preventing,neurodegenerative disorders such as Alzheimer's disease or Parkinson'sdiseases. They are also useful for treating invention also relates tothe use of said pharmaceutical compositions for treating proliferativedisorders such as cancers, or the proliferation of unicellular orpluricellular parasites. Other applications comprise the use of saidpharmaceutical compositions against cardiovascular disorders linked toproliferation. They also comprise their use for treating viralinjections (HIV, cytomegalovirus and herpes virus. The invention alsorelates to the use of said derivatives as herbicides.

Said pharmaceutical compositions can be administered in various formse.g. orally, topically, by injection (intravenously, subcutaneously,intraperitoneally, or rectally). They are more particularly administeredby the oral route.

For administration by the oral route, lozenges, compressed tablets,pills, tablets, capsules, drops, syrups, suspensions or emulsions, maybe used. These compositions advantageously comprise 100 to 1000 mg ofactive principle per dose unit, preferably 300 to 600 mg.

Other forms of administration include injectable solutions for theintravenous, subcutaneous or intramuscular route, formulated fromsterile or sterilizable solutions. They can also be suspensions oremulsions.

These injectable forms comprise 100 to 1000 mg of active principlepreferably 300 to 600 mg, per dose unit.

By way of indication, the dosage which can be used in a patient in needthereof corresponds to the following doses for example, 100 to 1000mg/day are thus administered to the patient 1 to 4 times per day for thetreatment of neurodegenerative disorders.

The invention also relates to biological reagents, the active principlesof which consist of the compounds of formula (I) as above-defined.

These reagents can be used as references or standards in studies of celldivision and phosphorylation mechanisms.

Other characteristics and advantages of the invention are given in theExamples disclosed hereinafter, with reference to FIGS. 1 to 7 whichrepresent, respectively;

FIG. 1: CDK1/cyclin B, CDK5/p25 and GSK-3β inhibition results withaloisines of the invention,

FIGS. 2A to 2C, enzyme activities for aloisine A in the presence of ATP(CD1/cyclin B: FIG. 2A; CDK5/p25: FIG. 2B, and GSK-3β: FIG. 2C),

FIG. 3, stereo view of the interactions between aloisine B and the CDK2ATP binding site,

FIGS. 4A to 4E, reversible inhibition of exponential cell growth byaloisine A, and

FIG. 5, the comparison of the effects of aloisine A on G0/G1 (A, B, C)and G2/M (D, E, F)

A/ Chemistry

Melting points were measured in open capillary tubes on anElectrothermal 9200 apparatus and are uncorrected. IR spectra were takenin KBr on an ATI Mattson genesis series FTIR. ¹H NMR were recorded on aVarian EM 360 A spectrometer (60 MHz) and chemical shifts (ppm) arereported relative to TMS. Signals are designated as follows: bs (broadsinglet), s (singlet), d (doublet), dd (doublet of doublets), t(triplet), m (multiplet). Mass spectra were determined on a LKB 209 (EIat 70 eV). Elemental microanalyses are indicated by the symbol of theelements and the results were within ±0.4% of the theoretical valuesunless otherwise stated; they were performed on a Perkin Elmer 240apparatus.

All experiments involving butyllithium or sodium hydride were carriedout in dried apparatus under an atmosphere of dry oxygen-free nitrogen.Tetrahydrofuran (THF) was distilled from benzophenone-sodium.Diisopropylamine and methyl-heterocycles were distilled and stored overbaryum oxide. Butyllithium (1.6M solution in hexane) was supplied byAcros and was assayed by titration against diphenylacetic acid. Alkyland aralkylpyrazines were prepared according to usual procedures. Gracesilicagel 60 A 20-45 μm was employed for column chromatographies.2-Phenylindole was purchased from Aldrich and used as received.

a—General Method for the Synthesis of Aloisines

Diisopropylamine (2.23 g; 0.022 mol) in THF (50 mL) was cooled to 0° C.,and butyllithium (0.022 mol) was added dropwise. After stirring 30 minat 0° C., the solution was cooled to −40° C. before, addition of thealkylpyrazine derivative (0.02 mol) in THF (20 mL). After 30 min, thenitrile derivative R6—CN (0.001 mol) in THF (20 mL) was added, and thesolution was stirred for 30 min at −40° C. and further (1 h-20 h) at 20°C., then hydrolyzed with a 10% aqueous solution of NH₄Cl. The organiclayer was dried over Na₂SO₄, and concentrated under vacuum. The crudeproduct was chromatographied on silicagel, eluted with methylenechloride, then ethyl acetate. If necessary the product was crystallizedfrom ethanol or methylene chloride-ethanol mixture.

b—Experimental Results

6-(2-Furyl)[5H]pyrrolo[2,3-b]pyrazine (1, RP19): mp 232.6° C.; IR 3157,3143, 3102 cm⁻¹; ¹H NMR δ (60 MHz, DMSO-d₆) 6.50-6.70 (m, 1H), 6.80 (s,1H), 7.05 (d, 1H, J=3 Hz), 7.80 (bs, 1H), 8.20 and 8.35 (2d, 1H each,J=3 Hz), 12.45 (bs, 1H). Anal. (C₁₀H₇N₃O) C, H, N.

6-(2-Thienyl)[5H]pyrrolo[2,3-b]pyrazine (2, RP6): mp 260.3° C.; IR 3208,3150, 3068 cm⁻¹; ¹H NMR δ (60 MHz, DMSO-d₆) 6.95 (bs, 1H), 7.15-7.45 (m,1H), 7.75 (s, 1H), 7.85 (bd, 1H), 8.25 and 8.45 (2d, 1H each, J=2.8 Hz),12.85 (bs, 1H). Anal. (C₁₀H₇N₃S) C, H, N.

6-(3-Thienyl) [5H]]pyrrolo(2,3-b]pyrazine (3, RP128): mp 230° C. dec; IR3095, 3050, 3000 cm⁻¹; ¹H NMR δ (60 MHz, DMSO-d₆) 6.95 (s, 1H),7.60-7.75 (m, 2H), 8.10-8.20 (m, 2H), 8.30 (d, 1H, J=2.6 Hz), 12.30 (bs,1H). Anal. (C₁₀H₇N₃S) C, H, N.

6-(2-Pyridyl)[5H]pyrrolo[2,3-b]pyrazine (4, RP13): mp 233.1° C.; IR3100, 3059 cm⁻¹; ¹H NMR δ (60 MHz, DMSO-d₆) 7.30-7.60 (m, 2H), 7.95-8.20(m, 2H), 8.30 and 8.45 (2d, 1H each, J=2.8 Hz), 8.75 (d, 1H, J=5 Hz),12.65 (bs, 1H). Anal. (C₁₁H₈N₄) C, H, N.

6-Phenyl[5H]pyrrolo[2,3-b]pyrazine (5, RP7): mp 216° C. (lit mp 215-216°C.); IR 3135, 3050 cm⁻¹; ¹H NMR δ (60 MHz, DMSO-d₆) 7.00 (s, 1H), 7.55(m, 3H), 7.90 (m, 2H), 8.25 and 8.50 (2d, 1H each, J=3 Hz), 11.90 (bs,1H); MS m/e 209 (M⁺) (100). Anal. (C₁₂H₉N₃) C, H, N.

6-(1-Naphtyl) [5H]pyrrolo[2,3-b]pyrazine (6, RP17): mp 216.4° C.; IR3214, 3110, 3048 cm⁻¹; ¹H NMR δ (60 MHz, DMSO-d₆) 6.95 (s, 1H),7.50-7.85 (m, 4H), 7.95-8.40 (m, 4H), 8.50 (d, 1H, J=2.5 Hz), 12.05 (bs,1H). Anal. (C₁₆H₁₁N₃) C, H, N.

3-Methyl-6-phenyl[5H]pyrrolo[2,3-b]pyrazine (8, RP18): mp 261.8° C.; IR3104, 3030, 2985, 2915, 2878, 2801 cm⁻¹; ¹H NMR δ (60 MHz, DMSO-d₆) 2.55(s, 3H), 7.15 (s, 1H), 7.35-7.60 (m, 3H), 7.75-8.10 (m, 2H), 8.40 (s,1H), 12.30 (bs, 1H); MS m/e 209 (M⁺) (100). Anal. (C₁₃H₁₁N₃) C, H, N.

6-[1-(4-Chlorophenyl)-1-cyclopropyl][5H]pyrrolo[2,3-b]pyrazine (9,RP124): mp 189.7° C.; IR 3210, 3125, 3049, 3000, 2940, 2850 cm⁻¹; ¹H NMRδ (60 MHz, DMSO-d₆) 1.50 (d, 4H), 6.15 (s, 1H), 7.30 (s, 4H), 8.10 and8.25 (2d, 1H each, J=3 Hz), 12.00 (bs, 1H). Anal. (C₁₅H₁₂N₃Cl) C, H, N.

6-(2-Methoxyphenyl)[5H]pyrrolo[2,3-b]pyrazine (10, RP9): mp 156.9° C.;IR 3080, 3051, 2925, 2887, 2830 cm⁻¹; ¹H NMR δ (60 MHz, DMSO-d₆) 3.90(s, 3H), 6.95-7.35 (m, 4H), 7.80-8.10 (m, 1H), 8.20 and 8.35 (2d, 1Heach, J=2.5 Hz), 11.90 (bs, 1H). Anal. (C₁₃H₁₁N₃O) C, H, N.

6-(3-Methoxyphenyl)[5H]pyrrolo[2,3-b]pyrazine (12, RP10): mp 195.7° C.;IR 3123, 2968, 2921, 2836 cm⁻¹; ¹H NMR δ (60 MHz, DMSO-d₆) 3.95 (s, 3H),6.90-7.80 (m, 5H), 8.25 and 8.40 (2d, 1H each, J=2.5 Hz), 12.55 (bs,1H). Anal. (C₁₃H₁₁N₃O) C, H, N.

6-(4-Methoxyphenyl)[5H]pyrrolo[2,3-b]pyrazine (14, RP11): mp 256.1° C.(lit. 238-240° C. dec.); IR 3143, 3035, 2959, 2857 cm⁻¹; ¹H NMR δ (60MHz, DMSO-d₆) 3.80 (s, 3H), 6.95-7.10 (m, 3H), 8.00 (d, 2H, J=8 Hz),8.15 and 8.35 (2d, 1H each, J=2.6 Hz), 12.35 (bs, 1H). Anal. (C₁₃H₁₁N₃O)C, H, N.

6-(3,5-Dimethoxyphenyl)[5H]pyrrolo[2,3-b]pyrazine (16, RP21): mp 216.7°C.; IR 3150, 2950, 2880 cm⁻¹; ¹H NMR δ (60 MHz, DMSO-d₆) 3.90 (s, 6H),6.55 (s, 1H), 7.20 (m, 3H), 8.15 and 8.35 (2d, 1H each, J=2.5 Hz), 12.40(bs, 1H). Anal. (C₁₄H₁₃N₃O₂) C, H, N.

6-(3,4,5-Trimethoxyphenyl)[5H]pyrrolo[2,3-b]pyrazine (17, RP16): mp231.7° C.; IR 3098, 2964, 2939, 2834 cm⁻¹; ¹H NMR δ (60 MHz, DMSO-d₆)3.75 (s, 3H), 3.95 (s, 6H), 7.25 (s, 1H), 7.40 (bs, 2H), 8.25 and 8.40(2d, 1H each, J=2 Hz), 12.45 (bs, 1H). Anal. (C₁₅H₁₅N₃O₃) C, H, N.

6-(4-Fluorophenyl)[5H]pyrrolo[2,3-b]pyrazine (18, RP76): mp 244° C. dec;IR 3149 cm⁻³; ¹H NMR δ (60 MHz, DMSO-d₆) 7.05-7.50 (m, 3H), 7.65-8.10(m, 2H), 8.20 and 8.35 (2d, 1H each, J=2.4 Hz), 12.45 (bs, 1H). Anal.(C₁₂H₈N₃F) C, H, N.

6-(4-Chlorophenyl)[5H]pyrrolo[2,3-b]pyrazine (19, RP14): mp 250° C. dec(lit. 250° C. dec.); IR 3300 cm⁻¹; ¹H NMR δ (60 MHz, DMSO-d₆) 7.20 (s,1H), 7.55 and 8.05 (2d, 2H each, J=8. Hz), 8.20 and 8.35 (2d, 1H each,J=2.4 Hz), 12.45 (bs, 1H). Anal. (C₁₂H₈N₃Cl) C, H, N.

6-(3,5-Dichlorophenyl)[5H]pyrrolo[2,3-b]pyrazine (20, RP15) : mp 252° C.dec; IR 3216, 3164, 3114 cm⁻¹; ¹H NMR δ (60 MHz, DMSO-d₆) 7.35 (s, 1H),7.75 (m, 1H), 8.15 (d, 2H), 8.25 and 8.40 (2d, 1H each, J=2.2 Hz), 12.40(bs, 1H). Anal. (C₁₂H₇N₃Cl₂) C, H, N.

6-(4-Bromophenyl) [5H]pyrrolo[2,3-b]pyrazine (21, RP77): mp 256° C. dec;IR 3211, 3109 cm⁻¹; ¹H NMR δ (60 MHz, DMSO-d₆) 7.20 (s, 1H), 7.70 and8.00 (2d, 2H each, J=8.2 Hz), 8.20 and 8.35 (2d, 1H each, J=2.5 Hz),12.45 (bs, 1H). Anal. (C₁₂H₈N₃Br) C, H, N.

6-(4-Trifluoromethylphenyl)[5H]pyrrolo[2,3-b]pyrazine(22, RP8): mp 238°C. dec; IR 3164 cm⁻¹; ¹H NMR δ (60 MHz, DMSO-d₆) 7.35 (s, 1H), 7.85 (d,2H, J=8.2 Hz), 8.10-8.50 (m, 4H), 12.70 (bs, 1H). Anal. (C₁₃H₈N₃F₃) C,H, N.

6-(4-Cyanophenyl) [5H]pyrrolo[2,3-b]pyrazine (23, RP20): mp 340° C. dec;IR 3464, 3056, 2205 cm⁻¹; ¹H NMR δ (60 MHz, DMSO-d₆) 7.45 (s, 1H),7.90-8.65 (m, 6H), 12.85 (bs, 1H). Anal. (C₁₃H₈N₄) C, H, N.

6-(4-Methylphenyl)[5H]pyrrolo[2,3-b]pyrazine (24, RP78): mp 265.4° C.;IR 3150, 3120, 2940, 2920 cm⁻¹; ¹H NMR δ (60 MHz, DMSO-d₆) 2.40 (s, 3H),7.05 (s, 1H), 7.30 and 7.90 (2d, 2H each, J=8 Hz), 8.15 and 8.30 (2d, 1Heach, J=2.4 Hz), 12.45 (bs, 1H). Anal. (C₁₃H₁₁N₃) C, H, N.

6-[4-(2-Dioxolyl)-phenyl][5H]pyrrolo[2,3-b]pyrazine (25, RP122): mp265.6° C.; IR 3120, 2980, 2889 cm⁻¹; ¹H NMR δ (60 MHz, DMSO-d₆) 3.95 (s,4H), 5.75 (s, 1H), 7.15 (s, 1H), 7.50 and 8.05, (2d, 2H each, J=7 Hz),8.20 and 8.35 (2d, 1H each, J=3 Hz), 12.45 (bs, 1H). Anal. (C₁₅H₁₃N₃O₂)C, H, N.

6-(4-Dimethylaminophenyl)[5H]pyrrolo[2,3-b]pyrazine (26, RP129): mp 271°C. dec; IR 3211, 3157, 2900, 2818, cm⁻¹; ¹H NMR δ (60 MHz, DMSO-d₆) 3.15(s, 6H), 6.80-6.90 (m, 3H), 7.85 (d, 2H, J=8.Hz), 8.05 and 8.20 (2d, 1Heach, J=3 Hz), 12.15 (bs, 1H). Anal. (C₁₄H₁₄N₄) C, H, N.

6-(4-Methoxyphenyl)-7-methyl[5H]pyrrolo[2,3-b]pyrazine (27, RP95): mp221.6° C.; IR 3142, 3043, 2955, 2844 cm⁻¹; ¹H NMR δ (60 MHz, DMSO-d₆)2.40 (s, 3H), 3.80 (s, 3H), 7.10 and 7.70 (2d, 2H each, J=7 Hz), 8.15and 8.30 (2d, 1H each, J=2.6 Hz), 12.00 (bs, 1H). Anal. (C₁₄H₁₃N₃O) C,H, N.

6-(3,4-Methoxyphenyl)-7-methyl[5H]pyrrolo[2,3-b]pyrazine (29, RP123): mp230.2° C.; IR 3102, 2963, 2920, 2850 cm⁻¹; ¹H NMR δ (60 MHz, DMSO-d₆)2.55 (s, 3H), 3.80 (s, 3H), 3.85 (s, 3H), 7.00-7.40 (m, 3H), 8.15 and8.30 (2d, 1H each, J=3 Hz), 12 (bs, 1H). Anal. (C₁₅H₁₅N₃O₂) C, H, N.

6-(4-Chlorophenyl)-7-methyl[5H]pyrrolo[2,3-b]pyrazine (30, RP80): mp260° C. dec.; IR 3148, 2920, 2853 cm⁻¹; ¹H NMR δ (60 MHz, DMSO-d₆) 2.40(s, 3H), 7.40-7.85 (m, 4H), 8.15 and 8.30 (2d, 1H each, J=2.5 Hz), 12.00(bs, 1H). Anal. (C₁₃H₁₀N₃Cl) C, H, N.

6-(4-Dimethylaminosulfamoyloxyphenyl)-7-methyl[5H]pyrrolo[2,3-b]pyrazine(31, RP125): mp 235.1° C.; IR 3140, 3045, 2970, 2925, 2880 cm⁻¹; ¹H NMRδ (60 MHz, DMSO-d₆) 2.45 (s, 3H), 2.95 (s, 6H), 7.45 and 7.90 (2d, 2Heach, J=8 Hz), 8.30-8.50 (m, 2H), 12.25 (bs, 1H). Anal. (C₁₅H₁₆N₄SO₃) C,H, N.

6-(4-Methoxyphenyl)-7-propyl[5H])pyrrolo[2,3-b]pyrazine (32, RP127): mp188.5° C.; IR 3215, 3158, 3055, 2958, 2934, 2866, 2836 cm⁻¹; ¹H NMR δ(60 MHz, DMSO-d₆) 0.9 (t, 3H, J=7 Hz), 1.70 (m, 2H), 2.80 (t, 2H, J=7Hz), 3.80 (s, 3H), 7.05 and 7.65 (2d, 2H each, J=8 Hz), 8.15 and 8.30(2d, 1H each, J=3 Hz), 12.00 (bs, 1H). Anal. (C₁₆H₁₇N₃O) C, H, N.

7-Allyl-6-(4-methoxyphenyl)[5H]pyrrolo[2,3-b]pyrazine (34, RP110): mp193.8° C.; IR 3135, 3063, 2962, 2934, 2878, 2838 cm⁻¹; ¹H NMR δ (60 MHz,CDCl₃) 3.65-3.85 (m, 2H), 3.90 (s, 3H), 4.80-5.20 (m, 2H), 5.75-6.45 (m,1H), 7.10 and 7.75 (2d, 2H each, J=8.2 Hz), 8.05 and 8.40 (2d, 1H each,J=2.4 Hz), 11.85 (bs, 1H). Anal. (C₁₆H₁₅N₃O) C, H, N.

7-(3-Chloropropyl)-6-(4-methoxyphenyl)[5H]pyrrolo[2,3-b]pyrazine (35,RP126): mp 178° C. dec; IR 3220, 3159, 3050, 3000, 2835 cm⁻¹; ¹H NMR δ(60 MHz, DMSO-d₆) 2.00-2.60 (m, 2H), 3.00 (m, 2H), 3.60 (t, 2H, J=6 Hz),3.80 (s, 3H), 7.10 and 7.70 (2d, 2H each, J=8 Hz). 8.15 and 8.35 (2d, 1Heach, J=3 Hz), 12.00 (bs, 1H). Anal. (C₁₆H₁₆N₃OCl) C, H, N.

7-Isopropyl-6-(4-methoxyphenyl)[5H]pyrrolo[2,3-b]pyrazine (36, RP102):mp 204.8° C.; IR 3135, 3050, 2957, 2924, 2859 cm⁻¹; H NMR δ (60 MHz,DMSO-d₆) 1.30 (d, 6H), 3.50 (m, 1H), 3.80 (s, 3H), 7.10 and 7.55 ( 2d,2H each, J=8 Hz), 8.15 and 8.35 (2d, 1H each, J=2.4 Hz), 11.75 (bs, 1H).Anal. (C₁₆H₁₇N₃O) C, H, N.

6-(4-Chlorophenyl)-7-isopropyl[5H]pyrrolo[2,3-b]pyrazine (37, RP90): mp208.6° C.; IR 3130, 3051, 2977, 2925, 2869 cm⁻¹; ¹H NMR δ (60 MHz,CDCl₃) 1.50 (d, 6H, J=6 Hz), 3.25 (m, 1H), 7.60 (s, 4H), 8.20 and 8.35(2d, 1H each, J=2.5 Hz), 12.00 (bs, 1H). Anal. (C₁₅H₁₄N₃Cl) C, H, N.

7-n-Butyl-6-(4-methoxyphenyl)[5H]pyrrolo[2,3-b]pyrazine (38, RP106): mp183.8° C.; IR 3143, 3050, 2956, 2934, 2870 cm⁻¹; ¹H NMR δ (60 MHz,DMSO-d₆) 1.00 (t, 3H, J=7.2 Hz), 1.60 (m, 4H), 3.00 (t, 2H, J=7.6 Hz),3.90 (s, 3H), 7.10 and 7.70 (2d, 2H each, J=8 Hz), 8.00 and 8.30 (2d, 1Heach, J=2.6 Hz), 11.75 (bs, 1H). Anal. (C₁₇H₁₉N₃O) C, H, N.

7-n-Butyl-6-(4-chlorophenyl)[5H]pyrrolo[2,3-b]pyrazine (40, RP108): mp200° C.; IR 3161, 3048, 2954, 2924, 2856 cm⁻¹; ¹H NMR δ (60 MHz,DMSO-d₆) 0.90 (t, 3H, J=6 Hz), 1.20-2.00 (m, 4H), 2.95 (t, 2H, J=7.2Hz), 7.65 (s, 4H), 8.25 and 8.40 (2d, 1H each, J=2.5 Hz), 12.05 (bs,1H). Anal. (C₁₆H₁₆N₃Cl) C, H, N.

7-n-Heptyl-6-(4-methoxyphenyl)[5H]pyrrolo[2,3-b]pyrazine (41, RP111): mp132.5° C.; IR 3142, 3064, 2955, 2925, 2850 cm⁻¹; ¹H NMR δ (60 MHz,CDCl₃) 0.90-2.00 (m, 13H), 3.05 (t, 2H, J=7.2 Hz), 3.90 (s, 3H), 7.05and 7.70 (2d, 2H each, J=8.2 Hz), 8.00 and 8.40 (2d, 1H each, J=3 Hz),12.05 (bs, 1H). Anal. (C₂₀H₂₅N₃O) C, H, N.

6-(4-Methoxyphenyl)-7-methylcyclopropyl[5H]pyrrolo[2,3-b]pyrazine (42,RP104): mp 193.9° C.; IR 3142, 3080, 3046, 3000, 2931, 2820 cm⁻¹; ¹H NMRδ (60 MHz, DMSO-d₆) 0.20-0.50 (m, 4H), 1.00-1.40 (m, 1H), 2.90 (d, 2H,J=6 Hz), 3.85 (s, 3H), 7.15 and 7.75 (2d, 2H each, J=8.2 Hz), 8.20 and8.40 (2d, 1H each, J=2.6 Hz), 12.20 (bs, 1H). Anal. (C₁₇H₁₇N₃O) C, H, N.

7-Benzyl-6-phenyl[5H]pyrrolo[2,3-b]pyrazine (44, RP92): mp 209.8° C.; IR3144, 3056, 3024, 2929, 2871 cm⁻¹; ¹H NMR δ (60 MHz, DMSO-d₆) 4.30 (s,2H), 7.20 (s, 6H), 7.75-8.25 (m, 4H), 8.30 and 8.40 (2d, 1H each, J=3Hz), 12.25 (bs, 1H). Anal. (C₁₉H₁₅N₃) C, H, N.

7-Benzyl-6-(4-chlorophenyl)[5H]pyrrolo[2,3-b]pyrazine (45, RP91): mp266.3° C.; IR 3138, 3050, 3025, 2928, 2858 cm⁻¹; ¹H NMR δ (60 MHz,DMSO-d₆) 4.25 (s, 2H), 7.15 (s, 5H), 7.55 (s, 4H), 8.20 and 8.35 (2d, 1Heach, J=3 Hz), 12.25 (bs, 1H). Anal. (C₁₉H₁₄N₃Cl) C, H, N.

6-(4-Methoxyphenyl)-7-methylcyclohexyl[5H]pyrrolo[2,3-b]pyrazine (46,RP98): mp 220.3° C.; IR 3434, 3135, 2921, 2850 cm⁻¹; ¹H NMR δ (60 MHz,DMSO-d₆) 0.85-1.80 (m, 11H), 2.80 (d, 2H, J=6.5 Hz), 3.80 (s, 3H), 7.15and 7.70 (2d, 2H each, J=8.2 Hz), 8.15 and 8.35 (2d, 1H each, J=2.5 Hz),11.90 (bs, 1H). Anal. (C₂₀H₂₃N₃O) C, H, N.

6-(4-Chlorophenyl)-7-methylcyclohexyl[5H]pyrrolo[2,3-b]pyrazine (47,RP99): mp 203.5° C.; IR 3142, 3048, 2928, 2847 cm⁻¹; ¹H NMR δ (60 MHz,DMSO-d₆) 0.80-1.75 (m, 11H), 2.80 (d, 2H, J=6.5 Hz), 7.65 (s, 4H), 8.20and 8.40 (2d, 1H each, J=2.4 Hz), 12.10 (bs, 1H). Anal. (C₁₉H₂₀N₃Cl) C,H, N.

c—General Method for the Demethylation of Methoxy-substituted6-phenyl[5H]pyrrolo[2,3-b]pyrazines

First, hydrobromic acid was redistilled over a trace of 50%hypophosphorus acid: 1 g for each 100 g of 48% hydrobromic acid. Methoxycompound (0.003 mol) was heated with hydrobromic acid (20 ml). Afterremoval of the aqueous forerun, the temperature reaches 126° C. The timerequired for demethylation varies from 3-10 h. The excess of hydrobromicacid was removed under reduced pressure, and the crude product wascrystallised from ethanol.

6-(2-Hydroxyphenyl)[5H]pyrrolo[2,3-b]pyrazine hydrobromide (11, RP109):mp 250° C. dec; IR 3419, 3354, 3090, 2710, 2641 cm⁻¹; ¹H NMR δ (60 MHz,DMSO-d₆) 5.80 (s, 3H), 6.80-7.30 (m, 4H), 7.70-8.00 (m, 1H), 8.40 (bs,3H), 12.85 (bs, 1H). Anal. (C₁₂H₉N₃O, HBr, H₂O) C, H, N.

6-(3-Hydroxyphenyl)[5H]pyrrolo[2,3-b]pyrazine hydrobromide (13, RP134):mp 258° C. dec.; IR 3448, 3137, 3085, 2700, 2630 cm⁻¹; ¹H NMR δ (60 MHz,DMSO-d₆) 6.70-7.50 (m, 7H), 8.65 (bs, 2H), 13.45 (bs, 1H). Anal.(C₁₂H₉N₃O, HBr) C, H, N.

6-(4-Hydroxyphenyl) [5H] pyrrolo[2,3-b]pyrazine hydrobromide (15, RP26):mp 255° C. dec; IR 3448, 3176, 3060 cm⁻¹; ¹H NMR δ (60 MHz, DMSO-d₆)6.65-6.85 (m, 4H), 7.70 and 7.85 (2d, 2H each, J=8.2 Hz), 8.15 and 8.25(2d, 1H each, J=3 Hz), 9.65 (s, 1H), 12.10 (bs, 1H). Anal. (C₁₂H₉N₃O,HBr, H₂O) C, H, N.

6-(4-Hydroxyphenyl)-7methyl[5H]pyrrolo[2,3-b]pyrazine hydrobromide (28,RP96): mp 262° C. dec;. IR 3465, 3143, 3090, 2796, 2759 cm⁻¹; ¹H NMR δ(60 MHz, DMSO-d₆) 2.45 (s, 3H); 7.00 and 7.70 (2d, 2H each, J=8.2 Hz),8.50 (bs, 2H), 9.80 (s, 2H), 13.00 (bs, 1H). Anal. (C₁₃H₁₁N₃O, HBr) C,H, N.

6-(4-Hydroxyphenyl)-7-propyl[5H]pyrrolo[2,3-b]pyrazine hydrobromide (33,RP132): mp 244° C. dec; IR 3187, 3100, 2965, 2873, 2798 cm⁻¹; ¹H NMR δ(60 MHz, DMSO-d₆) 0.85 (t, 3H, J=7 Hz), 1.35-1.90 (m, 2H), 3.10-2.75 (m,2H), 7.20 and 7.65 (2d, 2H each, J=8.2 Hz), 8.50 (s, 2H), 9.8 (s, 2H),13.1 (s, 1H). Anal. (C₁₅H₁₅N₃O, HBr) C, H, N.

7-n-Butyl-6-(4-hydroxyphenyl)[5H pyrrolo(2,3-b]pyrazine (39, RP107): mp281.4° C.; IR 3134, 3100, 2946, 2924, 2867 cm⁻¹; ¹H NMR δ (60 MHz,DMSO-d₆) 0.90 (t, 3H, J=7 Hz), 1.20-1.90 (m 4H), 2.90 (t, 2H, J=7.5 Hz),6.95 and 7.60 (2d, 2H each, J=7 Hz), 8.15 and 8.30 (2d, 1H each, J=2.6Hz), 9.80 (bs, 1H), 11.80 (bs, 1H). Anal. (C₁₆H₁₇N₃O) C, H, N.

6-(4-Hydroxyphenyl)-7-methylcyclopropyl[5H]pyrrolo[2,3-b]pyrazinehydrobromide (43, RP112) : mp 260° C. dec; IR 3482, 3335, 3064, 2983cm⁻¹; ¹H NMR δ (60 MHz, DMSO-d₆) 1.60 (d, 4H, J=6 Hz), 2.00-2.60 (m,3H), 2.90-3.60 (m, 4H), 7.05 and 7.90 (2d, 2H each, J=8.2 Hz ),8.55-8.80 (m, 2H), 13.45 (bs, 1H). Anal. (C₁₆H₁₅N₃O, HBr, H₂O) C, H, N.

B/ Biochemistry

Biochemical Reagents

Sodium ortho-vanadate, EGTA, EDTA, Mops, β-glycerophosphate,phenylphosphate, sodium fluoride, dithiothreitol (DTT),glutathione-agarose, glutathione, bovine serum albumin (BSA),nitrophenylphosphate, leupeptin, aprotinin, pepstatin, soybean trypsininhibitor, benzamidine, histone Hl (type III-S) were obtained from SigmaChemicals. [γ-³²P]-ATP (PB 168) was obtained from Amersham. The GS-1peptide has sequence SEQ ID N° 1 YRRAAVPPSPSLSRHSSPHQSpEDEEE.

Buffers

Homogenization Buffer: 60 mM β-glycerophosphate, 15 mMp-nitrophehylphosphate, 25 mM Mops (pH 7.2) , 15 mM EGTA, 15 mM MgCl₂, 1mM DTT, 1 mM sodium vanadate, 1 mM NaF, 1 mM phenylphosphate, 10 μgleupeptin/ml, 10 μg aprotinin/ml,10 μg soybean trypsin inhibitor/ml and100 μM benzamidine.

Buffer A: 10 mM MgCl₂, 1 mM EGTA, 1 mM DTT, 25 mM Tris-HCl pH 7.5, 50 μgheparin/ml.

Buffer C: homogenization buffer but 5 mM EGTA, no NaF and no proteaseinhibitors.

Tris-Buffered Saline -Tween-20 (TBST): 50 mM Tris pH 7.4, 150 mM NaCl,0.1% Tween-20.

Hypotonic Lysis Buffer (HLB): 50 mM Tris-HCl pH 7.4, 120 mM NaCl, 10%glycerol, 1% Nonidet-P40, 5 mM DTT, 1 mM EGTA, 20 mM NaF, 1 mMorthovanadate, 5 μM microcystin, 100 μg/ml each of leupeptin, aprotininand pepstatin.

Kinase Preparations and Assays

Kinases activities were assayed in Buffer A or C (unless otherwisestated), at 30° C., at a final ATP concentration of 15 μM. Blank valueswere subtracted and activities calculated as pmoles of phosphateincorporated for a 10 min. incubation. The activities are usuallyexpressed in % of the maximal activity, i.e. in the absence ofinhibitors. Controls were performed with appropriate dilutions ofdimethylsulfoxide. In a few cases phosphorylation of the substrate wasassessed by autoradiography after SDS-PAGE.

GSK-3α/β was either purified from porcine brain or expressed in andpurified from insect Sf9 cells. It was assayed, following a 1/100dilution in 1 mg BSA/ml 10 mM DTT, with 5 μl 40 μM GS-1 peptide as asubstrate, in buffer A, in the presence of 15 μM [γ-³²P] ATP (3,000Ci/mmol; 1 mCi/ml) in a final volume of 30 μl. After 30 min. incubationat 30° C., 25 μl aliquots of supernatant were spotted onto 2.5×3 cmpieces of Whatman P81 phosphocellulose paper, and, 20 sec. later, thefilters were washed five times (for at least 5 min. each time) in asolution of 10 ml phosphoric acid/liter of water. The wet filters werecounted in the presence of 1 ml ACS (Amersham) scintillation fluid.

CDK1/cyclin B was extracted in homogenisation buffer from M phasestarfish (Marthasterias glacialis) oocytes and purified by affinitychromatography on p9^(CKShs1)-sepharose beads, from which it was elutedby free p9^(CKShs1). The kinase activity was assayed in buffer C, with 1mg histone H1/ml, in the presence of 15 μM [λ-³²P] ATP (3,000 Ci/mmol; 1mCi/ml) in a final volume of 30 μl. After 10 min. incubation at 30° C.,25 μl aliquots of supernatant were spotted onto P81 phosphocellulosepapers and treated as described above.

CDK5/p25 was reconstituted by mixing equal amounts of recombinantmammalian CDK5 and p25 expressed in E. coli as GST(Glutathione-S-transferase) fusion proteins and purified by affinitychromatography on glutathione-agarose (p25 is a truncated version ofp35, the 35 kDa CDK5 activator). Its activity was assayed in buffer C asdescribed for CDK1/cyclin B.

Other kinases were expressed, purified and assayed as describedpreviously (9), (10).

Results

Inhibitory-Effects of Aloisines on CDK1, CDK5 & GSK-3

Kinases were assayed as above described in the presence of increasingconcentrations of aloisines. IC₅₀'s were calculated from thedose-response curves and are given in Table 1 hereinafter in μM.

TABLE 1

CDK1/ cyclin CDK5/ N^(o) a b c d 7 B p25 GSK-3  5 (RP7) — — — — — 5.004.00 2.30 10 (RP9) OCH₃ — — — — 20.00 23.00 3.30 11 (RP109) OH — — — —2.50 3.00 6.50 12 (RP10) — OCH₃ — — — 13.00 10.00 3.20 13 (RP134) — OH2.50 — — 14 (RP11) — — OCH₃ — — 2.00 4.00 1.10 15 (RP26) — — OH — — 1.201.00 1.20 16 (RP21) — OCH₃ — OCH₃ — 100.00 >100.00 60.00 17 (RP16) —OCH₃ OCH₃ OCH₃ — 100.00 >100.00 85.00 18 (RP76) — — F — — 2.30 1.00 1.9019 (RP14) — — Cl — — 1.80 — — 20 (RP15) — Cl — Cl— >100.00 >100.00 >100.00 21 (RP77) — — Br — — 4.00 >100.00 6.00 22(RP8) — — CF₃ — — 6.00 >100.00 7.20 24 (RP78) — — CH₃ — — 3.00 10.002.60 23 (RP20) — — CN — — 3.00 13.00 4.80 26 (RP129) — — N(CH₃)₂ — —20.00 >100.00 12.00 27 (RP95) — — OCH₃ — CH₃ 0.30 0.80 0.46 29 (RP123) —OCH₃— OCH₃ — CH₃ 1.10 1.00 2.00 32 (RP127) — — OCH₃ — (CH₂)₂CH₃ 0.400.50 0.40 35 (RP126) — — OCH₃ — (CH₂)₃—Cl 1.30 3.00 2.50 36 (RP102) — —OCH₃ — CH(CH₃)₂ 1.00 2.00 0.50 34 (RP110) — — OCH₃ — CH₂—CH═CH₂ 1.002.00 0.60 38 (RP106) — — OCH₃ — (CH₂)₃—CH₃ 0.70 1.50 0.92 41 (RP111) — —OCH₃ — (CH₂)₆—CH₃ 7.00 >100.00 >10.00 42 (RP104) — — OCH₃ — CH₂—C₃H₅1.00 >100.00 1.10 46 (RP98) — — OCH₃ — CH₂—C₆H₁₁ 5.00 >100.00 6.80 31(RP125) — — O—SO₂—N(CH₃)₂ — CH₃ 0.70 0.90 0.50 30 (RP80) — — Cl — CH₃0.40 5.00 1.70 37 (RP90) — — Cl — CH(CH₃)₂ 0.85 13.00 0.75 (aloisine B)40 (RP108) — — Cl — (CH₂)₃—CH₃ 0.20 >100.00 5.90 45 (RP91) — — Cl —CH₂—C₆H₅ 40.00 >100.00 6.80 44 (RP92) — — — — CH₂—C₆H₅ 2.00 >100.00 1.0047 (RP99) — — Cl — CH₂—C₆H₁₁ 10.00 >100.00 8.00 28 (RP96) — — OH — CH₃0.25 0.20 0.52 33 (RP132) — — OH — (CH₂)₂CH₃ 25.00 1.20 1.80 39 (RP107)— — OH — (CH₂)₃—CH₃ 0.15 0.20 0.65 (aloisine A) 43 (RP112) — — OH —CH₂—C₃H₅ 50.00 >100.00 3.00 Structure activity relationship ofaloisine-related compounds. CDK1/ cyclin CDK5/ N^(o) structure B p25GSK-3  1 (RP19)

12 9.30 15  2 (RP6)

7.00 2.00 1.20  3 (RP128)

2.30 1.00 0.80  4 (RP13)

21.00 53.00 15.00  6 (RP17)

>100.00 80.00 27.00  9 (RP124)

30.00 100.00 1.00 49 (RP130)

25.00 100.00 18.00 25 (RP122)

8.00 15.00 20.00  8 (RP18)

69.00 100.00 >100.00 48 (RP22)

100.00 7.00 >100.00

FIG. 1 gives the results obtained in the presence of increasingconcentrations of aloisines A and B. Activity is presented as % ofmaximal activity, i.e. measured in the absence of inhibitors.

Said results show that the aloisines of the invention are potentinhibitors of CDKs and GSK-3 and for most of them in the submicromolarrange.

Aloisine is a Competitive Inhibitor of ATP Binding

To investigate the mechanism of aloisine action, kinetic experimentswere performed by varying both ATP levels and aloisine A concentrations.Double reciprocal plots of kinetic data from assays of CDK1/cyclin B(A), CDK5/p25 (B) and GSK-3β (C) kinase activities at differentconcentrations of aloisine A are given on FIG. 2. Enzyme activities wereassayed as described in the Experimental section. ATP concentrations inthe reaction mixture varied from 0.1 to 0.25 mM (CDK1 and CDK5) or 0.015to 0.15 mM (GSK-3□). The concentrations of histone H1 (A, B) and GS-1(C) were kept constant at 0.7 mg/ml and 6.7 μM, respectively.

The data demonstrate that aloisine A acts as a competitive inhibitor forATP. These results are in complete agreement with the localization ofaloisine B within the ATP binding pocket of CDK2 (see below).

Crystallography

Expression, Purification and Crystallisation of Human CDK2

Human CDK2 was expressed from a recombinant baculo-virus in Sf9 insectcells and purified. Monomeric unphosphorylated CDK2 crystals were grownas previously described in (11).

X-ray Crystallography Data Collection and Processing

The CDK2-aloisine B dataset was collected from a monomeric CDK2 crystalsoaked for 60 h in 1 mM aloisine B in 1× mother liquor solution (50 mMammonium acetate, 10% PEG3350, 15 mM NaCl, 100 mM HEPES, pH7.4) plus 5%DMSO. Data was collected on beamline X-RAY DIFFRACTION at the ElettraLight Source at 100K after the crystal had been transferred briefly tocryo-protectant (mother liquor adjusted to contain 20% glycerol). Theimages were integrated with the MOSFLM package (12) and reflections weresubsequently scaled and merged using SCALA (13). Subsequent datareduction and structure refinement were pursued through programs of theCCP4 suite.

Aloisine B occupies the CDK2 ATP binding site and makes two hydrogenbonds to the CDK2 backbone within the hinge sequence that links the twolobes of the kinase. CDK2 is drawn in ribbon representation andcolour-ramped from blue through to red starting at the N-terminus. TheN-terminal lobe is dominated by a 5-stranded anti-parallel β-sheet andthe C-terminal lobe is predominantly α-helical. Aloisine B is drawn inball and stick mode bound at the ATP-binding site which lies in thecleft between the two domains. Aloisine B carbon atoms are colouredcyan, nitrogen atoms blue and the chlorine atom is drawn in yellow.

Unlike the natural ligand, ATP, aloisine B does not interact with thebackbone oxygen of Glu81, but instead accepts and donates a hydrogenbond respectively from the backbone nitrogen and oxygen atoms of Leu 83(FIG. 3) residues that lie within 4 Å of the bound aloisine B moleculeare drawn in ball and stick mode. Aloisine B carbon atoms are drawn incyan and those of CDK2 in green. Oxygen atoms are coloured red, nitrogenatoms are blue and the chlorine atom is drawn in yellow. Dotted linesrepresent hydrogen bonds (dO—>N or dN—O<3.4 Å) between aloisine B andthe backbone nitrogen and oxygen atoms of Leu 83. The Figure alsoincludes (2Fo-Fc)α-calc electron density for aloisine B calculated atthe end of refinement using map coefficients output from REFMAC withresolution between 20 and 1.9 Å. The map is contoured at a level of0.19e− Å⁻³ corresponding to 1.0 times the r.m.s. deviation of the mapfrom its mean value.

This hydrogen-bonding pattern has previously been observed in thestructures of monomeric CDK2 in complex with olomoucine, roscovitine,purvalanol B, OL567 and H717. The CDK2 ATP-binding site is tolerant of anumber of positions for the planar heterocyclic ring systems which are acharacteristic of the CDK inhibitors identified to date. The position ofthe aloisine B fused ring system within the CDK2 ATP binding site mostclosely resembles that of indirubin-5-sulphonate and oxindole-3.However, being smaller than indirubin-5-sulphonate and in a differentorientation to oxindole-3, aloisine B does not fill the back of theATP-binding cleft and form an equivalent edge-to-ring stackinginteraction with the side-chain of Phe 80.

Aloisines, Kinase Selectivity

Aloisine A, the most active aloisine so far, was tested for selectivityon 26 highly purified kinases. Kinase activities were assayed withappropriate substrates (for example histone H1, casein, myelin basicprotein and peptides), with 15 μM ATP and in the presence of increasingconcentrations of aloisine A. IC₅₀ values were estimated from thedose-response curves and are presented in Table 3.

TABLE 3 Kinase inhibition selectivity of aloisine A. Protein KinasesIC₅₀ (μM) CDK1/cyclin B 0.15 CDK2/cyclin A 0.12 CDK2/cyclin E 0.40CDK4/cyclin D1 >100.00 CDK5/p35 0.16 erk1 18.00 erk2 22.00 c-raf >100.00MAPKK >100.00 c-Jun N-terminal kinase 3.3-10 protein kinase C α >100.00protein kinase C β1 >100.00 protein kinase C β2 >100.00 protein kinase Cγ >100.00 protein kinase C δ >100.00 protein kinase C ε >100.00 proteinkinase C η >100.00 protein kinase C ζ >100.00 cAMP-dependent proteinkinase 100.00 cGMP-dependent protein kinase 100.00 GSK3-α 0.50 GSK3-β1.50 Casein kinase 1 >100.00 Casein kinase 2 >100.00 Insulin receptortyrosine kinase 60.00 PIM 1 >10.00

Most kinases tested were poorly or not inhibited (IC₅₀>10 μM). However,two families of kinases, GSK-3α/β and CDKs were strongly sensitive toaloisine A (IC₅₀'s of 0.65 and 0.15 μM, respectively) (FIG. 1; Table 3).Among the CDKs, CDK1, CDK2 and CDK5, but not CDK4 were inhibited byaloisine A. This is reminiscent of other CDK inhibitors, such aspurines, hymenialdisine, paullones, and indirubins, which inhibitCDK1/2/5 but have much less effect on or not CDK4/6. Although aloisinesappear to be remarkably specific to CDKs and GSK-3, the actual spectrumof their intracellular targets remains to be identified. For thispurpose we are currently designing an immobilized aloisine matrix topurify aloisine-binding proteins by the affinity chromatography methoddescribed for purines and paullones.

C/ Cell Biology

Reagents

Penicillin, streptomycin, nocodazole, insulin, transferrin,progesterone, putrescine, sodium selenite,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT),RNAse A, propidium iodide were purchased from Sigma.

Cell Cultures

Clonal human NT2 teraterocarcinoma cells were obtained from Stratagene(La Jolla, Calif.) and grown in Dulbecco's Modified Eagle Medium:Nutrient Mixture F-12 with 2 mM L-Glutamine (BIO WHITTAKER) supplementedwith 5% FCS and containing penicillin (20 UI/ml) and streptomycin (20μg/ml) at 37° C., in a humidified atmosphere containing 5% CO₂ in air.

NT2 Differentiation

Differentiation of NT2 cells to hNT cells was induced according to themethod of Pleasure et al. (17) modified by Soulié et al. Briefly, afterthe second replating, cells were cultured in serum-free medium with acombination of mitotic inhibitors (1 μM cytosine arabinoside, 10 μmfluorodeoxyuridine and 10 μM uridine) and a mixture of salt and hormones(25 μg insulin /ml, 100 μg transferrin/ml, 20 nM progesterone, 60 μMputrescine and 30 nM sodium selenite) for 5 days before treatment.

Treatment with Aloisine

Exponentially growing cells were incubated for 24 h with aloisine A(stock solution dissolved in dimethylsulfoxide). Nocodazole treatment ofcells was performed at a concentration of 0.04 μg nocodazole/ml ofmedium for 24 h. Following the nocodazole treatment, cells were washedtwice with fresh medium and cultured with or without aloisine A for 24h. To perform serum deprivation, cells were maintained in serum-freemedium for 40 h. Following serum deprivation, cells were washed twiceand cultured in fresh serum-containing medium with or without aloisine Afor 40 h.

Cell Viability Assay

To quantify the toxicity of aloisine A on NT2 cells and hNT humanneurons, the inhibition of cellular reduction of MTT to MTT formazan wasmeasured according to Saillé et al. Following aloisine A exposure, cellswere incubated with 0.5 mg MTT/ml fresh medium at 37° C. for 1 hour. Theformazan products were dissolved in DMSO and quantified by measurementof the absorbance at 562 nm.

Cell Cycle Analysis by Flow Cytometry

Cells were trypsinised, collected by centrifugation and fixed in cold70% ethanol for at least 4 h. Fixed cells were washed in PBS, incubatedwith 10 μg RNAse A/ml and stained with 25 μg propidium iodide/ml for 1 hat 37° C. The stained cells were then analysed for cell cycledistribution on a FACSort flow cytometer (Becton Dickinson). Cell cycleanalyses were performed multiCYCLE (18).

The effect of aloisine A on the cell cycle distribution was investigatedfor NT2 cells by flow cytometry. Unsynchronised cells (FIG. 4E) wereexposed to 20 μM aloisine A for 40 h (FIG. 4F). The proliferation arrestinduced by aloisine A in exponentially growing cells was clearlyaccompanied by an accumulation in G2/M phase. No signs of apoptosis weredetectable, confirming the lack of apparent toxicity observed before.

The effects of aloisine A on NT2 cells synchronized either in G0/G1 byserum deprivation or in G2/M by nocodazole treatment was alsoinvestigated. The results are given on FIG. 4: the cell cycle phasedistribution was analysed by flow cytometry following propidium iodidestaining. (A, B, C). NT2 cells were synchronized by serum deprivationfor 24 h (A), then cultured for 40 h in fresh medium without (B) or with20 μM aloisine A (C). (D, E, F). NT2 cells were synchronized bynocodazole treatment (0.2 μg/ml) for 24 h (D), then cultured for 40 h infresh medium without (E) or with 20 μM aloisine A (F).

Serum deprivation for 24 h lead to a significant increase of cells inG0/G1 (FIG. 5A). Cells were then re-exposed to a serum-enriched mediafor 40 h in the absence (FIG. 5B) or presence (FIG. 5C) of 20 μMaloisine A. Aloisine A-treated cells remained essentially in G0/G1, witha small additional accumulation of G2/M cells, most probably derivedfrom the initial S phase sub-population (FIG. 5C). Control cellsredistributed in a classical cell cycle pattern (FIG. 5B). Nocodazoletreatment for 24 h lead to a massive accumulation of cells in G2/M (FIG.5D). Cells were then washed to remove nocodazole and incubated for 40 hin the absence (FIG. 5E) or presence (FIG. 5F) of 20 μM aloisine A. 40 hafter nocodazole withdrawal, control cells redistributed in the variouscell cycle phases (FIG. 8E). In contrast, the majority of cells exposedto aloisine A after nocodazole treatment remained in G2/M (FIG. 5F),precluding the increase in G1/G0 seen in control cells. A smallsub-G2-peak may indicate a minor onset of apoptotic cell death.

All together these data indicate that aloisine A has anti-proliferativeproperties and that it is able to block both the exit from G0/G1 and theexit from G2/M, suggesting the existence of several intracellulartargets. A G1 arrest correlates with aloisine A's high potency againstCDK2/cyclin E. The inability to enter S phase might also be result frominhibition of GSK-3, a kinase known to be involved in cyclin Dldegradation. The G2/M arrest correlates well with the potency ofaloisine A against CDK1/cyclin B.

REFERENCES

-   1. Meijer, L. Cyclin-dependent kinases inhibitors as potential    anticancer, anti-neurodegenerative, anti-viral and anti-parasitic    agents. Drug Resistance Update 2000, 3, 83-88.-   2. Toogood, P. L. Cyclin-dependent kinase inhibitors for treating    cancer. Med. Res. Rev. 2001, 21, 487-498.-   3. Gray, N.; Détivaud, L.; Doerig, C.; Meijer, L. ATP-site directed    inhibitors of cyclin-dependent kinases. Curr. Med. Chem. 1999, 6,    859-876.-   4. Fischer, P. M.; Lane, D. P. Inhibitors of cyclin-dependent    kinases as anti-cancer therapeutics. Curr. Med. Chem. 2000, 7,    1213-1245.-   5. Pestell, R.; Mani, S.; Wange, C.; Wu, K.; Francis, R.    Cyclin-dependent kinase inhibitors: novel anticancer agents. Expert    Opin. Investig. Drugs 2000, 9, 1849-1870.-   6. Rosiana, G. R.; Chang, Y. T. Targeting hyperproliferative    disorders with cyclin dependent kinase inhibitors. Exp. Opin. Ther.    Patents 2000, 10, 1-16.-   7. Sielecki, T. M.; Boylan, J. F.; Benfield, P. A.; Trainor, G. L.    Cyclin-dependent kinase inhibitors: useful targets in cell cycle    regulation. J. Med. Chem. 2000, 43, 1-18.-   8. Kaubisch, A.; Schwartz G. K. Cyclin-dependent kinase and protein    kinase C inhibitors: a novel class of antineoplastic agents in    clinical development. Cancer J. 2000, 6, 192-212.-   9. Meijer, L.; Borgne, A.; Mulner, O.; Chong, J. P. J.; Blow, J. J.;    Inagaki, N.; Inagaki, M.; Delcros, J. G.; Moulinoux, J. P.    Biochemical and cellular effects of roscovitine, a potent and    selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and    cdk5. Eur. J. Biochem. 1997, 243, 527-536.-   10. Meijer, L.; Thunissen, A. M. W. H.; White, A.; Garnier, M.;    Nikolic, M.; Tsai, L. H.; Walter, J.; Cleverley, K. E.; Salinas, P.    C.; Wu, Y. Z.; Biernat, J.; Mandelkow, E. M.; Kim, S.-H.;    Pettit, G. R. Inhibition of cyclin-dependent kinases, GSK-3β and    casein kinase 1 by hymenialdisine, a marine sponge constituent.    Chem. & Biol. 2000, 7-51-63.-   11. Lawrie, A. M.; Noble, M. E.; Tunnah, P.; Brown, N. R.;    Johnson, L. N.; Endicott, J. A. Protein kinase inhibition by    staurosporine revealed in details of the molecular interaction with    CDK2. Nature Structural Biol. 1997, 4, 796-800.-   12. Leslie, A. G. W. Joint CCP4 and ESF-EAMCB Newsletter on Protein    Crystallography. 1992, vol. 26.-   13. CCP4. The CCP4 (Collaborative Computational Project Number 4)    suite: Programs for protein crystallography. Acta Cryst. D, 1994,    50, 760-763.-   14. Navaza, J. AMoRe: An automated package for molecular    replacement. Acta. Cryst., 1994, A50, 157-163.-   15. Murshudov, G. N.; Vagin, A. A.; and Dodson, E. J. Refinement of    macromolecular structures by the maximum-likelihood method. Acta    Cryst. D, 1997, 53, 240-255.-   16. Jones, T. A., Zou, J. Y., Cowan, S. W., Kjeldgaard, M. Improved    methods for building protein models in electron density maps and the    location of errors in these models. Acta Cryst. A, 1991, 47,    110-119.-   17. Pleasure, S. J.; Page, C.; Lee, V. M. Y. Pure, post-mitotic    polarized human neurons derived from NT2 cells provide a system for    expressing exogenous proteins in terminally differentiated    neurons. J. Neurosci. 1992, 12, 1802-1815.-   18. Damiens, E.; Baratte, B.; Marie, D.; Eisenbrand, G.; and    Meijer, L. Anti-mitotic properties of indirubin-3′-monoxime, a    CDK/GSK-3 inhibitor: induction of endoreplication following prophase    arrest. Oncogene, 2001, 20, 3786-3797.

1. Pyrrolo [2,3b]-pyrazine derivatives having the general formula (I):

wherein: R2 and R3 are identical or different, and represent H, C1-C6alkyl, said alkyl being a straight or branched-chain alkyl, which can besubstituted, R6 is an optionally substituted aromatic cycle Ar or acycloalkyl, said cycloalkyl being optionally substituted by an arylgroup which can also be substituted, R7 is H, C1-C6 alkyl,(alk.)_(n)-hal., CH₂—CH═CH₂, CH₂-cycloalkyl, CH₂-Ar, with “alk.” being aC1-C6 alkylene group, n being 1-6, Z is H or CH₃.
 2. The pyrrolo[2,3b]-pyrazine derivatives of claim 1, wherein Ar is phenyl, naphtyl,furyl, thienyl, pyridyl, cyclopropyl phenyl, phenyl dioxolyl.
 3. Thepyrrolo[2,3b]-pyrazine derivatives of claim 1, wherein the Cycloalkylgroup is a C3-C6 cycloalkyl.
 4. The pyrrolo[2,3b]-pyrazine derivativesof claim 1, wherein the substitutions groups are selected in the groupcomprising one or more halogen (F, Cl, Br, I, CF₃), OH, NH₂, N(H,alkyl), N(alkyl)₂, O-alkyl, COOH, COO-alkyl, CONH₂, CON(H, alkyl),CON(alkyl)₂, NHCONH₂, NHCON (H, alkyl), NHCON (alkyl)₂, N(alkyl)CONH₂,N(alkyl)CON(H,alkyl), N(alkyl)CON(alkyl)₂, alkoxy, CN, O—SO₂—NH₂,O—SO₂—N (H, alkyl), —O—SO₂—N (alkyl)₂, SH,S-alkyl.
 5. Thepyrrolo[2,3b]-pyrazine derivatives of claim 1, with an IC₅₀≦10 μM withrespect to CDK1/cyclin B and/or CDK5/p25 and/or GSK-3 and having formula(II):

wherein: the phenyl group at position 6 is substituted by one, two orthree R substituents selected in the group comprising: H, —OH, alkyl, —Oalkyl, hal., —NH₂, —N(H,alkyl), —N(alkyl)₂, —O—SO₂—NH₂, —O—SO₂—N (H,alkyl), —O—SO₂—N(alkyl)₂, —COOH, —COO-alkyl, CONH₂, —CON(H,alkyl),—CON(alkyl)₂, R7 is H, alkyl, (alk.)_(n) hal., —CH₂—CH═CH₂,(alk.)_(n)-cycloalkyl, alk.-Ar, and Z is H or CH₃.
 6. Thepyrrolo[2,3b]-pyrazine derivatives of claim 5, corresponding to thederivatives of formula (II) wherein R=H, OH, alkoxy, hal., alkyl andR7=H or to derivatives wherein R=alkoxy, and R7=alkyl, (alk.)_(n)-hal.,CH₂—CH═CH₂, or R=O—SO₂—N-(alkyl)₂, hal., OH, R7=alkyl, n=1-3 and Z=H. 7.The pyrrolo[2,3b]-pyrazine derivatives of claim 5, having an IC₅₀value≦5 μM with respect to CDK1/cyclin B, CDK5/p25 and GSK-3.
 8. Thepyrrolo[2,3b]-pyrazine derivatives of claim 7, corresponding to thederivatives of formula (II) wherein R=H, p-alkoxy, p- and m-alkoxy,p-OH, p-hal., p-alkyl, p-O—SO₂—N (alkyl)₂, R7 is alkyl, (alk.)_(n)-hal.,CH₂—CH═CH₂, or H, Z is H, and n=1-3.
 9. The pyrrolo[2,3b]-pyrazinederivatives of claim 8, corresponding to compounds wherein (a-ecorrespond to the position of R on the phenyl group): the phenyl groupis unsubstituted and R7 is H, or Ra, Rb and Rd=H, Rc=alkoxy, OH or hal.,and R7=H, or Ra, Rb and Rd=H, Rc=alkoxy and R7=alkyl, or Ra and Rd=H, Rband Rc=alkoxy and R7=alkyl, or Ra, Rb and Rd=H, Rc=alkoxy and R7=alkyl,or Ra, Rb and Rd=H, Rc=alkoxy, OH, hal. and R7=alkyl, (alk.)_(n)-hal,CH₂—CH═CH₂, or Ra, Rb and Rd=H, Rc=OH, R7=alkyl.
 10. Thepyrrolo[2,3b]-pyrazine derivatives of claim 5 having an IC₅₀≦1 μM withrespect to CDK1/cyclin B, CDK5/p25 and GSK-3.
 11. Thepyrrolo[2,3b]-pyrazine derivatives of claim 10, corresponding toderivatives of formula (II) wherein R is p-alkoxy, p-O—SO₂—N-(alkyl)₂,p-OH and R7 is alkyl.
 12. The pyrrolo[2,3b]-pyrazine derivatives ofclaim 11, corresponding to compounds with Ra, Rb and Rd=H, Rc=alkoxy,O—SO₂—N(alkyl)₂, or OH and R7=alkyl.
 13. The pyrrolo[2,3b]-pyrazinederivatives of claim 5, having an IC₅₀≦0.5 μM with respect toCDK1/cyclin B, CDK5/p25 and GSK-3, wherein Ra, Rb and Rd=H, Rc=alkoxy orOH and R7=alkyl.
 14. The pyrrolo[2,3b]-pyrazine derivatives of claim 1,has an IC₅₀ value≦10 μM with respect to CDK1/cyclin B and CDK5/p25 orGSK-3, or to CDK5/p25 and GSK-3.
 15. The pyrrolo[2,3b]-pyrazinederivatives of claim 14, having an IC₅₀≦10 μM with respect to CDK5/p25and GSK-3.
 16. The pyrrolo[2,3b]-pyrazine derivatives of claim 15,wherein R=H, OH, alkoxy, hal., alkyl, O—SO₂—N(alkyl)₂, and R7=H, alkyl,(alk.)_(n)-hal., CH₂CH═CH₂.
 17. The pyrrolo[2,3b]-pyrazine derivativesof claim 15, having an IC₅₀ value≦5 μM with respect to CDK5/p25 andGSK-3, with R is H, p-alkoxy, OH, hal., O—SO₂—N-(alkyl)₂ and R7 is H,alkyl, (alk.)_(n), hal., CH₂—CH═CH₂.
 18. The pyrrolo[2,3b]-pyrazinederivatives of claim 17, having Ra, Rb, Rc, Rd and R7=H, or Ra, Rb andRd=H, Rc=alkoxy, hal., (alk.)_(n)-hal., or OH and R7=H, or Ra, Rb andRd=H, Rc=alkoxy or OSO₂—N(alkyl)₂, hal., OH and R7=alkyl, or Ra andRd=H, Rb and Rc=alkoxy and R7=alkyl.
 19. The pyrrolo[2,3b]-pyrazinederivatives of claim 15, having an IC₅₀ value≦1 μM with respect toCDK5/p25 and GSK-3, with R=p-alkoxy, p- and m-dialkoxy, hal.,p-O—SO₂—N(alkyl)₂, p-OH and R7=H or alkyl.
 20. Thepyrrolo[2,3b]-pyrazine derivatives of claim 19, herein Ra, Rb, Rd=H,Rc=alkoxy and R7=alkyl, or Ra and Rd=H, Rb and Rc=alkoxy and R7=alkyl,or Ra, Rb and Rd=H, Rc=O—SO₂—N(alkyl)₂ or OH and R7=alkyl.
 21. Thepyrrolo[2,3b]-pyrazine derivatives of claim 15, having an IC₅₀≦0.5 μMwith respect to CDK5/p25 and GSK-3, with Ra, Rb, and Rd=H, Rc=alkoxy orOH, and R7=alkyl.
 22. The pyrrolo[2,3b)-pyrazine derivatives of claim14, wherein said derivatives have an IC₅₀≦10 μM with respect to CDK1 andGSK3, with R=H, OH, alkoxy, hal., alkyl, CN, O—SO₂—N(alkyl)₂ and R7=H,alkyl, (alk.)_(n)-hal, CH₂—CH═CH₂, alk.-cycloalkyl, alk.-aryl.
 23. Thepyrrolo[2,3b]-pyrazine derivatives of claim 22, wherein said derivativeshave an IC₅₀≦5 μM with respect to CDK1 and GSK-3, with R=H, p-alkoxy, p-and m-alkoxy, p-OH, p-hal., p-O—SO₂-N(alkyl)₂, p-CN, and R7=H or alkyl,(alk.)_(n) hal., CH₂—CH═CH₂, (alk.)_(n)-cycloalkyl, (alk.)_(n)-aryl. 24.The pyrrolo[2,3b]-pyrazine derivatives of claim 23, wherein saidderivatives have Ra, Rb and Rd=H, Rc=alkoxy, OH, hal., alkyl, CN andR7=H, or Ra, Rb, Rd=H, Rc=alkoxy and R7=alkyl, (alk.)_(n)-hal. orCH₂—CH═CH₂, or Ra and Rd=H, Rb and Rc=alkoxy and R7=alkyl, or Ra, Rb andRc=H, Rd=O—SO₂—N-(alkyl)₂, and R7=alkyl, or Ra, Rb and Rd=H, Rc=hal. andR7=(alk.)_(n)-aryl.
 25. The pyrrolo[2,3b]-pyrazine derivatives of claim22, having an IC₅₀ value≦1 μM with respect to CDK1 and GSK-3, withR=p-alkoxy, p-O—SO₂—N(alkyl)₂, p-hal., H, p-OH, R7=alkyl, or(alk.)_(n)-hal, CH₂—CH═CH₂, (alk.)_(n)-cycloalkyl, (alk.)_(n)-aryl. 26.The pyrrolo[2,3b]-pyrazine derivatives of claim 25, wherein Ra, Rb andRd=H, Rc=alkoxy, OH, O—SO₂—N(alkyl)₂, hal. and R7=alkyl, CH₂—CH═CH₂,CH₂-cycloalkyl.
 27. The pyrrolo[2,3b]-pyrazine derivatives of claim 22,wherein said derivatives have an IC₅₀ value≦0.5 μM with respect toCDK1/cyclin B and GSK-3, with Ra, Rb and Rd=H, Rc=alkoxy or OH andR7=alkyl.
 28. The pyrrolo[2,3b]-pyrazine derivatives of claim 14,wherein said derivatives have an IC₅₀≦10 μM with respect to CDK1/cyclinB and CDK5/p25, with R=H, OH, alkoxy, hal., alkyl, O—SO₂—N(alkyl)₂ andR7=H, alkyl, (alk.)_(n)-hal., CH₂—CH═CH₂.
 29. The pyrrolo[2,3b]-pyrazinederivatives of claim 28, having an IC₅₀≦5 μM with respect to CDK1/cyclinB and GSK-3, and R is preferably H, O-alkoxy, p-alkoxy, m- and p-alkoxy,p-OH., p-hal., p-O—SO₂—N(alkyl)₂ and R7 is H, alkyl, (alk.)_(n)-hal.,CH₂—CH═CH₂.
 30. The pyrrolo[2,3b]-pyrazine derivatives of claim 29,wherein Ra, Rb, Rc, Rd and R7=H, or Ra=OH and Rb, Rc, Rd and R7=H, orRc, Rb and Rd=H, Rc=alkoxy, OH or hal. and R7=H, (alk.)_(n)-hal.,CH₂—CH═CH₂, alkyl, or Ra and Rd=H, Rb and Rc=alkoxy and R7=H, or Ra, Rband Rd=H, Rc=O—SO₂—N-(alkyl)₂ or hal. and R7=alkyl.
 31. ThePyrrolo[2,3b]-pyrazine derivatives of claim 28, wherein said derivativeshave an IC₅₀≦1 μM with respect to CDK1/cyclin B and GSK-3, andR=p-alkoxy, p-O—SO₂-N(alkyl)₂, p-hal., p-OH and R7=alkyl.
 32. Thepyrrolo[2,3b]-pyrazine derivatives of claim 31, wherein Ra, Rb and Rd=H,Rc=alkoxy OH or O—SO₂—N(alkyl)₂ and R7=alkyl.
 33. Thepyrrolo(2,3b]-pyrazine derivatives of claim 28, wherein said derivativeshave an IC₅₀≦0.5 μM with respect to CDK1/cyclin B and GSK-3, and Ra, Rb,and Rd=H, Rc=alkoxy or OH and R7=alkyl.
 34. The pyrrolo[2,3b]-pyrazinederivatives of claim 1, with an IC₅₀≦10 μM with respect to CDK1/cyclinB, CDK5 and GSK-3 has formula (III), and even ≦5 μM with respect toCDK5/p25 and GSK-3.


35. The pyrrolo[2,3b]-pyrazine derivatives of claim 1, having formula IVwith an IC₅₀≦5 μM with respect to CDK1/cyclin B, CDK5/p25 and GSK-3, andan IC₅₀ value≦1 μM with respect to CDK5/p25 and GSK-3.


36. The pyrrolo[2,3b]-pyrazine derivatives of claim 1 wherein R2 and R3,and/or Z and/or R7 are different from H.
 37. A method for preparing thepyrrolo[2,3b]-pyrazine derivatives of formula I according to claim 1comprising reacting alkyl-pyrazines of formula (V)

wherein: R1 and R3 are as above defined, and Alkyl is a C1-C6 alkyl,with aromatic nitrites, R6CN, wherein R6 is as above defined. 38.Pharmaceutical compositions comprising an effective amount of at leastone derivative of claim 1 as active principle, in association with apharmaceutically acceptable carrier.
 39. The pharmaceutical compositionsof claim 38 for treating or preventing neurodegenerative disorders suchas Alzheimer' s disease and Parkinson' s diseases.
 40. Thepharmaceutical composition of claim 38 for treating anti proliferativedisorders such as, but not limited to, cancers. This includes, but isnot limited to, the use against the proliferation of unicellular orpluricellular parasites, the use against cardiovascular disorders linkedto proliferation, the use as herbicides.
 41. The pharmaceuticalcompositions of claim 38, administered in various forms e.g. orally,topically, by injection (intravenously, subcutaneously,intraperitoneally, or rectally).
 42. The pharmaceutical composition ofclaim 41, for administration by the oral route comprising 100 to 1000 mgof active principle per dose unit, preferably 300 to 600 mg.
 43. Thepharmaceutical compositions of claim 41 under injectable forms,comprising 100 to 1000 mg of active principle preferably 300 to 600 mg,per dose unit.